1 Introduction to Fermentation

Processes
1.1 Historical Highlights of Fermentation Processes
By 2012, industrial Microbiology touched the major sectors that define human
activity: food, fuel, and health. The history of biotechnology starts with bread-
making, utilizing yeast, about 8000 years ago (Table 1.1). Fermentation of grains
and fruits to alcoholic beverages was carried out in Egypt and other parts of the
ancient world in about 2500 bc. Other types of food fermentation practiced for
thousands of years include the transformation of milk into cheeses and fermentation
of soybeans. However, it was not until 1857 that Pasteur proved that alcoholic
fermentation was caused by living cells, namely, yeasts. In the ensuing 100 years,
the intentional manipulation of microbial fermentations to obtain food products,
solvents, and beverages, and later, substances having therapeutic value as
antibiotics gave rise to a large fermentation industr.

Traditional fermentation processes, such as those involved in the production of

fermented dairy products and alcoholic beverages, have been performed for
thousands of years. However, it is less than 150 years ago that the scientific basis of
these processes was first examined.

The birth of Microbial Biotechnology or Industrial Microbiology largely began with the
studies of Pasteur. In 1857 he finally demonstrated beyond doubt that alcoholic
fermentation in beer and wine production was the result of microbial activity,
rather than being a chemical process. Pasteur also noted that certain organisms
could spoil beer and wine, and that some fermentations were aerobic, whereas
others were anaerobic. He went on to devise the process of pasteurization, a major
contribution to food and beverage preservation, which was originally developed to
preserve wine. In fact, many of the early advances of both pure and applied
microbiology were through studies on beer brewing and wine making. Pasteur’s
publications and others were important catalysts for the progress of industrial
fermentation processes.

Of the further advances that followed, none were more important than the
development of pure culture techniques by Hansen at the Carlsberg Brewery in
Denmark. Pure strain brewing was carried out here for the first time in 1883, using a

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yeast isolated by Hansen, referred to as Carlsberg Yeast No. 1 (Saccharomyces
carlsbergensis, now classified as a strain of Saccharomyces cerevisiae).

During the early part of the 20 th century, further progress in this field was relatively
slow. Around the turn of the century there had been major advancements in the
large-scale treatment of sewage, enabling significant improvement of public health
in urban communities.

However, the first novel industrial-scale fermentation process to be introduced was

the acetone–butanol fermentation, developed by Weizmann (1915) using the
bacterium Clostridium acetobutylicum.

In the early 1920s an industrial fermentation process was also introduced for the
manufacture of citric acid, employing a filamentous fungus (mould), Aspergillus
niger.

Further innovations in fermentation technology were greatly accelerated in the 1940s

through efforts to produce the antibiotic penicillin, stimulated by the vital need for
this drug during World War II. Not only did production rapidly move from small-scale
surface culture to large-scale submerged fermentations, but it led to great advances
in both media and microbial strain development. The knowledge acquired had a
great impact on the successful development of many other fermentation industries.

More recent progress includes the ability to produce monoclonal antibodies for
analytical, diagnostic, therapeutic and purification purposes, pioneered by Milstein
and Kohler in the early 1970s.

However, many of the greatest advances have followed the massive developments in
genetic engineering (recombinant DNA technology) over the last 20 years. This
technology has had, and will continue to have, a tremendous influence on traditional,
established and novel fermentation processes and products. It allows genes to be
transferred from one organism to another and allows new approaches to strain
improvement. The basis of gene transfer is the insertion of a specific gene sequence
from a donor organism, via an expression vector, into a suitable host. Hosts for
expression vectors can be prokaryotes such as the bacterium Escherichia coli;
alternatively, where post-translational processing is required, as with some human
proteins, a eukaryotic host is usually required, e.g., a yeast.

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1.2 Fermentation Processes
1.2.1 Definition of Fermentation
The term 'fermentation' is derived from the Latin verb ‘fervere’ to ‘boil’, thus
describing the appearance of the action of yeast on extracts of fruit or malted grain.
The boiling appearance is due to the production of carbon dioxide bubbles caused by
the anaerobic catabolism of the sugar present in the extract. However, fermentation
has come to have different meanings to biochemists and to industrial microbiologists.
Its biochemical meaning relates to the generation of energy by the catabolism of
organic compounds, whereas its meaning in industrial microbiology tends to be much
broader.

The production of alcohol by the action of yeast on malt or fruit extracts has been
carried out on a large scale for very many years and was the first 'industrial' process
for the production of a microbial metabolite. Thus, industrial microbiologists have
extended the term fermentation to describe any process for the production of
product by the mass culture of a microorganism. Brewing and the production of
organic solvents may be described as fermentations in both senses of the word but
the description of an aerobic process as a fermentation is obviously using the term in
the broader, microbiological context.

There are five major groups of commercially important fermentations:

1. Those that produce microbial cells (or biomass) as the product.
2. Those that produce microbial enzymes.
3. Those that produce microbial metabolites.
4. Those that produce recombinant products.
5. Those that modify a compound which is added to the fermentation the
transformation process.

1.2.2 The Components of a Fermentation Process

Regardless of the type of fermentation (with the possible exception of some
transformation processes) an established process may be divided into six basic
component parts:
1. The formulation of media to be used in culturing the process organism during the
development of the inoculum and in the production fermenter.
2. The sterilization of the medium, fermenters and ancillary equipment.

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3. The production of an active, pure culture in sufficient quantity to inoculate the
production vessel.
4. The growth of the organism in the production fermenter under optimum
conditions for product formation.
5. The extraction of the product and its purification.
6. The disposal of effluents produced by the process.

1.2.3 Advantages of Microorganisms in Industrial Processes

A vast range of important products, many of which were formerly manufactured by
chemical processes, are now most economically produced by microbial fermentation
and biotransformation processes. Microorganisms also provide valuable services.
They have proved to be particularly useful because of (i) the ease of their mass
cultivation, (ii) speed of growth, (iii) use of cheap substrates that in many cases are
wastes, and (iv) the diversity of potential products. In addition, (v) their ability to
readily undergo genetic manipulation has opened up almost limitless possibilities for
new products and services from the fermentation industries.

1.3 Typical Operations of a Fermentation Process

A typical operation involves both upstream processing (USP) and downstream
processing (DSP) stages (Figure 1.1).

1.3.1 Upstream Processing (USP)

The USP is associated with all factors and processes leading to and including the
fermentation, and consists of three main areas: (1) The producer microorganism, (2)
The fermentation medium, and (3) The fermentation.

1.3.1.1 The Producer Microorganism

Key factors relating to this aspect are: the strategy for initially obtaining a suitable
industrial microorganism, strain improvement to enhance productivity and yield,
maintenance of strain purity, preparation of a reliable inoculum and the continuing
development of selected strains to improve the economic efficiency of the process.
For example, the production of stable mutant strains that vastly overproduce the
target compound is often essential.

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Some microbial products are primary metabolites, produced during active growth
(the trophophase), which include amino acids, organic acids, vitamins and
industrial solvents such as alcohols and acetone. However, many of the most
important industrial products are secondary metabolites, which are not essential for
growth, e.g. alkaloids and antibiotics. These compounds are produced in the
stationary phase of a batch culture, after microbial biomass production has peaked
(the idiophase).

Figure 1.1: An outline of upstream and downstream processing operations.

1.3.1.2 The Fermentation Medium

The selection of suitable cost-effective carbon and energy sources, and other
essential nutrients, along with overall media optimization are vital aspects of process
development to ensure maximization of yield and profit. In many instances, the basis
of industrial media are waste products from other industrial processes, notably sugar
processing wastes, lignocellulosic wastes, cheese whey and corn steep liquor.

1.3.1.3 The Fermentation

Industrial microorganisms are normally cultivated under rigorously controlled
conditions developed to optimize the growth of the organism or production of a
target microbial product. The synthesis of microbial metabolites is usually tightly
regulated by the microbial cell. Consequently, in order to obtain high yields, the
environmental conditions that trigger regulatory mechanisms, particularly repression
and feedback inhibition, must be avoided.

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Fermentations are performed in large fermenters often with capacities of several
thousand litres. These range from simple tanks, which may be stirred or unstirred, to
complex integrated systems involving varying levels of computer control. The
fermenter and associated pipe-work, etc., must be constructed of materials, usually
stainless steel, that can be repeatedly sterilized and that will not react adversely with
the microorganisms or with the target products. The mode of fermenter operation
(batch, fed-batch or continuous systems), the method of its aeration and agitation,
where necessary, and the approach taken to process scale-up have major influences
on fermentation performance.

1.3.2 Downstream Processing (DSP)

Conventional DSP includes all unit processes that follow fermentation. They involve
(i) cell harvesting, (ii) cell disruption, (iii) product purification from cell extracts or
the growth medium, and (iv) finishing steps. However, attempts are now being made
to integrate fermentation with DSP operations, which often increases process
productivity. Overall, DSP must employ rapid and efficient methods for the
purification of the product, while maintaining it in a stable form. This is especially
important where products are unstable in the impure form or subject to undesirable
modifications if not purified rapidly. For some products, especially enzymes,
retention of their biological activity is vital. Finally, there must be safe and
inexpensive disposal of all waste products generated during the process.

DSP will usually involve more than one stage. Downstream processing costs (as
approximate proportions of selling prices) of fermentation products vary
considerably, e.g., with yeast biomass, penicillin G and certain enzymes, processing
costs as percentages of selling prices are 20%, 20–30% and 60–70% respectively.

1.4 Fermentation Methods

1.4.1 Shake-Flask Fermentation
Aerated and anaerobic fermentations at the laboratory scale are typically carried out
in shaken (not stirred) flasks in an incubator-shaker. There are many different types
available, with a model based on temperature-controlled, heated air, etc. The air
serves to both heat and cool the shake fl asks that are agitated by an orbital motion
of the shaker table (Figure 1.2). The flasks, nutrient medium, and caps are
sterilized at 121°C for 30 min in an autoclave. Wet heat (steam) is needed to obtain

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effective killing of microorganisms and spores that might be found in the flask or
nutrient solution.

Figure 1.2: Schematic diagram of incubator-shaker used for shake flask culture of
microbial cells.

Shake-flask studies are used early in the development of an industrial fermentation

since multiple nutrient compositions and different cultures can be evaluated in
parallel studies, in the same incubator. The pH in a shake-flask is controlled using
buffer, while other nutritional factors are controlled by the makeup of the broth.

The temperature range over which many types of organisms will grow are relatively
narrow and will typically fall within 20–60°C and at atmospheric pressure.
Microorganisms that are psychotrophs require lower temperatures, while
archaebacteria (Archaea) require higher temperatures (up to 90°C) and sometimes,
elevated pressure to grow. In this case, special incubation chambers are needed. The
circulation of air in the incubator also serves to remove heat of fermentation. The
oxygen requirements for aerobic microorganisms are met by the swirling action of
the shake fl ask, accompanied by diffusion of air through the porous, sterile, cotton
plug that is fitted on top of the flask.

The culturing of strict anaerobes [i.e., microorganisms that are killed by oxygen in
the ppb (parts per billion) range] requires that inoculation of the microorganism be
carried out in a special hood, known as an anaerobic chamber, so that oxygen is
excluded and prevents oxygen from dissolving in the broth. This enclosed chamber

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resembles a glove-box. Traces of oxygen are scavenged from the N 2 gas by passing
the gas over hot (200–300°C) copper filings that react with trace oxygen to form
copper oxides, and this oxygen-free nitrogen is used to fill the chamber.

1.4.2 Controlled Laboratory Fermentations in Sealed Glass

Vessels
Controlled laboratory fermentations at a larger scale are typically carried out in
sealed glass vessels. Volumes of these vessels range from 1 to 20-litre, with the
working volume (broth) typically being about 70% of the fermenter volume.

At the laboratory, scale, the entire vessel, including head-plates and fermentation
media, can be placed into an autoclave for sterilization. Such a fermenter contains
sealed ports for pH and O2 probes, as well as ports for addition of acid or base for
purposes of pH control. Aeration of the fermentation broth is carried out by bubbling
compressed air that has been sterile-filtered through a 0.2-µm filter, into the bottom
of the vessel. The 0.2-µm cutoff of such a filter is small enough to block any airborne
spores or microorganisms from entering the fermenter with the air. Ports enable
samples to be taken for intermittent analysis by chromatography, mass
spectroscopy, or other methods. These data are then used to generate graphs of the
fermentation time-course. Heating and cooling of the fermenter is achieved by
passing thermostat water through the cooling coil (i.e., hollow baffle) or a jacket.

1.4.3 Industrial-Scale Fermentation

Industrial-scale fermentation for manufacture of high-value products is carried out in
large, 316 stainless-steel vessels. The principle on which this equipment is based is
similar to that of the laboratory-scale fermenter although numerous and complex
mixing, aeration, and scale-up issues must be addressed. Sterilization is carried out
in place, and special designs are needed to minimize the risk of holdover of small
amounts of liquid that could serve as reservoirs of microbial contamination from one
batch to the next. Polished surfaces minimize the occurrence of small pores or
pockets in the metal that might retain microbial spores. Layout of the piping must
ensure that there are no low spots where liquid could pool and be a source of
contamination.

Unlike a chemical reactor, a biological reactor contains living entities that respond to
externally applied changes in their environment by generating new reaction

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pathways, new enzymes, and new products. These changes are propagated and
copied through cell division in a manner that is neither possible nor observed in
chemical reactors that contained a fixed amount of a defined catalyst.

1.5 Fermentation Products

The overall economics of fermentation processes are influenced by the costs of raw
materials and consumables, utilities, labour and maintenance, along with fixed
charges, working capital charges, factory overheads and operating outlay.

Fermentation products can be broadly divided into two categories: (i) high volume,
low value products, or (ii) low volume, high value products. Examples of the first
category include most food and beverage fermentation products, whereas many fine
chemicals and pharmaceuticals are in the latter category.

1.5.1 Food, Beverages, Food Additives and Supplements

A wide range of fermented foods and beverages have been produced throughout
recorded history. They continue to be major fermentation products worldwide and
are of vast economic importance. Fermented dairy products, for example, result
from the activities of lactic acid bacteria in milk, which modify flavour and texture,
and increase long-term product stability. Yeasts are exploited in the production of
alcoholic beverages, notably beer and wine, due to their ability to ferment sugars,
derived from various plant sources, to ethanol. Most processes use strains of one
species, Saccharomyces cerevisiae, and other strains of this yeast are used as
baker’s yeast for bread dough production.

Several organic acids derived from microbial action are employed in food
manufacture and for a wide range of other purposes. The first human use was for
acetic acid, as vinegar, produced as a result of the oxidation of alcoholic beverages
by acetic acid bacteria. A further aerobic fermentation involves citric acid
production by the filamentous fungus, Aspergillus niger, which has become a major
industrial fermentation product, as it has numerous food and non-food applications.

Also, most of the amino acids and vitamins used as supplements in human food
and animal feed are produced most economically by microorganisms, particularly if
high-yielding over-producing strains are developed. In addition, some
microorganisms contain high levels of protein with good nutritional characteristics

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suitable for both human and animal consumption. This so-called ‘single-cell
protein’ (SCP) can be produced from a wide range of microorganisms cultivated on
low-cost carbon sources.

1.5.2 Health-Care Products

In terms of providing human benefit, antibiotics are probably the most important
compounds produced by industrial microorganisms. Most are secondary metabolites
synthesized by filamentous fungi and bacteria, particularly the actinomycetes. Well
over 4,000 antibiotics have now been isolated, but only about 50 are used regularly
in antimicrobial chemotherapy. The best known and probably the most medically
useful antibiotics are the -lactams, penicillins and cephalosporins, along with
aminoglycosides (e.g., streptomycin) and the tetracyclines. New antibiotics are still
being sought as resistance to established antibiotics has become a major problem in
recent years, through the misuse and overuse of these drugs.
Other important pharmaceutical products derived from microbial fermentation and/or
biotransformations are alkaloids, steroids and vaccines. More recently,
therapeutic recombinant human proteins such as insulin, interferons and human
growth hormone have been produced by a range of microorganisms. This is a
rapidly expanding field and many more recombinant therapeutic products are likely
to come on to the market over the coming years.

1.5.3 Microbial Enzymes

Microbial enzymes, particularly extracellular hydrolytic enzymes, have numerous
roles as process aids or in the production of a wide range of specific food and non-
food products. Proteases, for instance, are extensively used as additives to washing
powders, in the removal of protein hazes from beer and as microbial rennets for the
production of cheese. Several carbohydrases are employed in the production of a
diversity of sugar syrups from starch. For example, high-fructose corn syrup is
produced by hydrolysing corn starch to glucose using -amylase and
amyloglucosidase, and the resulting glucose is then isomerized to a sweeter
molecule, fructose, by a glucose isomerase. All of these examples involve the use of
‘bulk’ enzymes. Smaller quantities of highly purified ‘fine’ enzymes are used for
numerous specialized purposes.

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Immobilization of enzymes or whole cells, by their attachment to inert polymeric
supports, allows easier recovery and reuse of the biocatalyst, and some enzymes are
much more stable in this form. Also, the product does not become contaminated with
the enzyme. Applications of immobilized biological catalysts include the production of
amino acids, organic acids and sugar syrups.

1.5.4 Industrial Chemicals and Fuels

Industrial feedstock chemicals supplied through fermentation include various
alcohols, solvents such as acetone, organic acids, polysaccharides, lipids and raw
materials for the production of plastics. Some of these fermentation products also
have applications in food manufacture.

Fossil fuels, especially oil, are likely to become exhausted within the next 50–100
years, resulting in the need to develop alternative sources of energy. Biological fuel
generation may make an increasing contribution, particularly in the conversion of
renewable plant biomass to liquid and gaseous fuels. This plant biomass can be in
the form of cultivated energy crops, natural vegetation, and agricultural, industrial
and domestic organic wastes. Currently, methane and ethanol are the main
products, although other potential fuels can be generated using microorganisms,
including hydrogen, ethane, propane and butanol.

1.5.5 Environmental Roles of Microorganisms

Microorganisms are particularly important in waste-water treatment, which
utilizes the metabolic activities of diverse mixed microbial populations capable of
degrading any compound that may be presented to them. The two main objectives
are to destroy all pathogenic microbes present in the sewage, particularly the causal
organisms of the water-borne diseases cholera, dysentery and typhoid. The second
objective is to break down the organic matter in waste-water to mostly methane and
carbon dioxide, thereby producing a final effluent (outflow) that can be safely
discharged into the environment.

Microbial activities can also be employed in the degradation of man-made

xenobiotic compounds within waste streams and in the bioremediation of
environments contaminated by these materials.

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Microbial-based ‘clean technology’ is also being increasingly used in the
desulphurization of fuels and the leaching of metals (e.g., copper, iron, uranium
and zinc) from low-grade mineral ores and wastes using species of Thiobacillus and
Sulfolobus. Environmental biological control is a further area where microorganisms
are employed in an effort to reduce our reliance on synthetic chemical pesticides.

Bacteria, fungi, protozoa and viruses are cultivated to produce biomass or cell
products for the biocontrol of fungal, insect and nematode pests of agricultural
crops, along with some vectors of human and animal diseases.

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